Method of detecting native proBNP

ABSTRACT

The present invention relates to antibodies specifically binding to native proBNP, a method for specific detection of native proBNP, a method of correlating the level of native proBNP to the diagnosis of heart failure, a kit for detection of native proBNP and to a hybridoma cell line producing an antibody to native proBNP.

RELATED APPLICATIONS

This application is a continuation of PCT/EP2004/005092 filed May 12,2004, which claims priority to EP 03010591.0 filed May 12, 2003.

FIELD OF THE INVENTION

The present invention relates to antibodies specifically binding to asubpopulation of total proBNP termed native proBNP, a method forspecific detection of native proBNP, a method of correlating the levelof native proBNP to the diagnosis of heart failure, a kit for detectionof native proBNP and to a hybridoma cell line producing an antibody tonative proBNP.

BACKGROUND OF THE INVENTION

Heart failure is a widespread phenomenon, especially in the westernworld. According to the Roche medical dictionary (Urban & Schwarzenberg,1993), heart failure is the acute or chronic inability of the heart togenerate the blood flow required for metabolism during exercise or evenat rest or to assure the venous reflux (backward and forward failure).Thus the pump function of the heart is weak. The causes of heart failureare very complex. Among others, inflammatory and degenerativemodifications of the cardiac muscle, coronary perfusion disorder,coronary infarction and injuries are to be mentioned here. This leads tomodifications of the peripheral bloodstream, disorders of the breathingsystem, renal function, and electrolyte metabolism (edema,) and to areduced performance of the muscular system of the skeleton.

According to the New York Heart Association (NYHA), heart failure isdivided into the following NYHA classes using physical tests aftereffort: I means completely free from pain after normal physical effort,II means low limitation of the physical toughness, III means stronglimitation of the physical toughness, IV means that with each physicalactivity, the insufficiency symptoms increase which most of the timealso exist at rest.

For an effective medicament treatment of heart failure by means ofglycosides, vasodilators, ACE inhibitors, and/or β-blockers, it is firstof all necessary to exactly and correctly identify and diagnose heartfailure, to classify it, if possible, according to the degree ofseverity, and to additionally monitor the course of treatment.

In the art, some serum markers are discussed as marker candidates for anearly diagnosis of heart failure as, for example, ANP (atrialnatriuretic peptid) hormone and proANP, CNP (C-natriuretic peptide),adrenomedullin, neuropeptide Y, endotheline, and BNP (brain natriureticpeptide). ANP and proANP theoretically would represent suitable markersfor the diagnosis of heart failure; in practice they are, however, notvery stable or only have a short half life in blood, which represents aserious drawback to routine diagnostic measurements (Buckley, M. G., etal., Clin. Sci. 95 (1998) 235-239; Cleland, J. G., et al., Heart 75(1996) 410-413).

A frequently cited and meaningful marker is BNP (brain natriureticpeptide). Originally, BNP was identified in the brain of pigs. It is acardiac hormone which structurally and functionally resembles ANP(Sudoh, T., et al., Nature 332 (1988) 78-81). Human BNP, consisting of32 amino acids, is mainly secreted by the heart ventricles andcirculates in the human blood plasma. The use of BNP as a diagnosticmarker is, for example, known from EP 542,255. BNP has an intramoleculardisulfide bridge and is not a very stable analyte. This presumably isdue to its physiological function as a hormone that must be broken downquickly. Therefore, its use as a diagnostic marker requires careful andspecial attention in sample collection and processing (Masuta, C., etal., Clin. Chem. 44 (1998) 130; Tsuji, T., et al., Clin. Chem. 40 (1994)672-673).

The precursor molecule of BNP, i.e., proBNP, consists of 108 aminoacids. proBNP is cleaved into the aforementioned 32 C-terminal aminoacids (77-108) called BNP and the N-terminal amino acids 1-76 calledN-terminal proBNP (or NT-proBNP). BNP, N-terminal proBNP (1-76) as wellas further breakdown products (Hunt, P. J., et al., Biochem. Biophys.Res. Com. 214 (1995) 1175-1183) circulate in blood. Whether the completeprecursor molecule (proBNP 1-108) also occurs in the plasma is notcompletely resolved. It is, however, described (Hunt, P. J., et al,Peptides, Vol. 18, No. 10 (1997), 1475-1481) that a low release ofproBNP (1-108) in plasma is detectable but that, due to the very quickpartial breakdown at the N-terminal end, some amino acids are absent.

As known from the art, the N-terminal proBNP (1-76) is considered amarker of heart failure.

WO 93/24531 and U.S. Pat. No. 5,786,163 describe an immunological methodof identifying N-terminal proBNP and the antibodies used for it. In WO93/24531, polyclonal antibodies (PAB's) against one single peptidederived from the N-terminal proBNP are produced. It is shown that theantibodies produced bind to the immunization peptide (amino acids 47-64)in a competitive test format.

In the competitive test performed in WO 93/24531, the peptide 47-64 in alabelled form competes as a tracer with proBNP in a sample or theunlabelled peptide standard 47-64 for binding to polyclonal antibodiesfrom rabbit serum. Only a moderate competition is reached after 48 hoursof incubation, resulting in a lower limit of detection of approximately250 fmol/ml. The long incubation times of this competitive test are notacceptable for routine measurements of the samples in automatedlaboratories.

Hunt, P. J., et al., Clinical Endocrinology 47 (1997) 287-296, alsodescribe a competitive test for the detection of N-terminal proBNP. Inthis assay, a complex extraction of the plasma sample is necessarybefore the measurement can be performed; this may lead to thedestruction of the analyte and erroneous measurements. The antiserumused is produced analogously to WO 93/24531 by immunization with asynthetic peptide. Hunt et al. produce the antiserum by immunizationwith the N-terminal proBNP amino acids I-13, and a peptide consisting ofamino acids 1-21 is used as a standard. For this test, long incubationtimes are necessary, too. After an incubation of 24 hours, a lowerdetection limit of 1.3 fmol/ml is reached.

Ng, L., et al., WO 00/35951 describe a further method for determiningN-terminal proBNP. This method is based on use of antibodies raisedagainst a synthetic peptide corresponding to amino acids 65 to 76 ofhuman proBNP.

Hughes, D., et al., Clin. Sci. 96 (1999) 373-380, report on twodifferent assays for N-terminal proBNP. In a first assay, a polyclonalantibody generated with an immunogen comprising a synthetic peptidecorresponding to amino acids 65-76 of proBNP is used, whereas in asecond assay, the polyclonal antibody was generated in analogy but toamino acids 37-49. According to the data produced by Hughes, D., et al.,an antibody generated and reactive with the peptide corresponding toamino acids 37-49 of proBNP does not react with intact endogenousproBNP. An assay based thereon does not discriminate patients with leftventricular dysfunction from normal controls. With the assay based onproBNP 65-76, the same patient groups could be clearly discriminated.

Goetze, J. P., et al., Clin. Chem. 48 (2002) 1035-1042, describe anassay for the most N-terminal amino acids (1-21) of N-terminal proBNP.Their assay is based on a polyclonal antibody raised against a syntheticpeptide corresponding to the same amino acids (1-21) of proBNP.

The assay of Goetze, J. P., et al., supra, requires complete digestionof the sample and the various proBNP's comprised therein. It is saidthat this assay also was efficient in reduction of non-specific binding.

Karl, J., et al., WO 00/45176, for the first time show that sensitiveand rapid detection of N-terminal proBNP is possible in a sandwichimmunoassay. Preferred epitopes, as described in WO 00/45176, arebetween amino acids 10 and 50 of N-terminal proBNP.

US 2003/0219734 refers to the fact that a plurality of differentBNP-related polypeptides derived from proBNP (1-108), BNP (77-108), aswell as from N-terminal proBNP (1-76) may be present in a sample.

Mair, J., et al., Clin. Chem. Lab. Med. 39 (2001) 571-588, havesummarized the impact of cardiac natriuretic peptide determination onthe diagnosis and management of heart failure. They stress thatcurrently available commercial assays are not standardized, i.e., theyhave not been calibrated against common standards. In some assays evenan extraction of plasma is needed. Consequently, the results obtainedwith assays from different manufacturers may differ markedly. Therefore,reference intervals and decision limits derived from clinical studiesare only valid for the particular assay used and must not beextrapolated to other assays for N-terminal proBNP.

Along the same lines, Goetze, J. P., et al., supra and Mair, J., Clin.Chem. 48 (2002) 977-978, note that the discrepancies between differentassays of N-terminal proBNP, both with respect to the values obtained aswell as with regard to their clinical implications, represent a crucialproblem to the widespread use of this marker candidate.

Obviously a great need exists to provide for an improved, e.g., morereproducible, better standardized, better characterized, and moreclinically relevant assay for N-terminal proBNP.

It was a task of the present invention to develop a more specific assayfor measurement of N-terminal proBNP and/or a clinically relevantfragment or subpopulation of N-terminal proBNP.

The invention as described below and claimed in the appending claims atleast partially solves one or more of the problems known in the art.

SUMMARY OF THE INVENTION

It has surprisingly been found that it is possible to specificallydetect a subpopulation of all proBNP species (total proBNP) present inthe circulation. This subpopulation is termed “native N-terminal proBNP”or simply “native proBNP”. Strikingly, it appears that the subpopulationof native proBNP is clinically more relevant as compared to the totalproBNP.

In a first embodiment, the present invention relates to an isolatedantibody specifically binding to native proBNP.

The invention also relates to a method for specific detection of nativeproBNP comprising the steps of contacting a sample suspected or known tocontain proBNP with an antibody specifically binding to native proBNPunder conditions allowing for the formation of an antibody to nativeproBNP-native proBNP complex and detecting the complex formed.

Further, the invention discloses a method for diagnosing heart failurecomprising detection of native proBNP and correlating the level ofnative proBNP to heart failure, whereby this correlated value of nativeproBNP is indicative for the absence, the presence, or the status ofheart failure.

The invention also relates to a kit for measurement of native proBNPcomprising an antibody specifically binding to native proBNP andauxiliary reagents for detection of native proBNP. Also claimed aremonoclonal antibodies specific for native proBNP as produced byhybridoma cell lines MAB 1.21.3 and MAB 16.1.39, respectively, whichhave been deposited with the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ).

DESCRIPTION OF THE DRAWINGS

FIG. 1: Epitope identification for MAB 1.21.3. The reactivity profile ofMAB 1.21.3 has been analyzed by use of 69 different biotinylated 8-merpeptides derived from the sequence of proBNP (1-76), each shifted by oneamino acid thus covering the complete sequence of proBNP (1-76).Extinction is given in mE-units. Strong reactivity has been found topeptides numbers 39 to 42.

FIG. 2: Instrument settings used in the BIACORE analyses. Thespecificity for native proBNP of the various antibodies to proBNP hasbeen assessed using the mode of operation of the BIACORE 3000 analyzer(Biacore AB, Sweded) as given in this figure.

FIGS. 3 to 7: Correlation of MAB 1.21.3 to various mono- and polyclonalanti-proBNP antibodies. Twenty human sera with a concentration of proBNPof about 10 mg/ml and above (as determined by using MAB 1.21.3 andsynthetic proBNP as a calibrator) have been analyzed in a sandwich assayusing the BIACORE 3000 analyzer. Values measured with MAB 1.21.3 aregiven on the x-axis. The corresponding values determined with theantibody used in the method comparison are given in the y-axis.Correlations of MAB 1.21.3 to MAB 18.4.34, MAB 18.29.23, PAB 30-38, PAB44-51 and PAB 41-46 are given in FIGS. 3, 4, 5, 6, and 7, respectively.

DESCRIPTION OF THE INVENTION

During the course of our experiments leading to the present invention,it has been found and established that at least two populations ofproBNP exist in human blood. One population of proBNP appears torepresent the majority of all proBNP molecules, which can be detected byimmunological methods. This population is termed “total” proBNP. Ourstudies have shown that the proBNP molecules which may be summarized astotal proBNP apparently have a central core structure in common whichranges from about amino acid position 10 to about amino acid position 66of proBNP. Preferably the total proBNP detected by a method according tothe present invention comprises amino acids 10 through 66. As theskilled artisan will appreciate, such total proBNP can be easilydetected by immunological procedures either in a competitive or in asandwich assay format. Preferably a sandwich assay is used to detecttotal proBNP. Such sandwich assay may be designed to comprise antibodiesbinding C- and N-terminal to the native proBNP epitope, respectively.However, it is also possible to, e.g., detect total proBNP usingantibodies capable of sandwich formation and both reactive with epitopesN-terminal to the native proBNP epitope. Obviously, in such competitiveor in such sandwich determination of total proBNP, an antibody to nativeproBNP must not be used.

The term “native proBNP” denotes any proBNP molecule on which theepitope as recognized by MAB 1.21.3 is present. According to thefindings demonstrated further below, this epitope is only present on asubpopulation of all proBNP-molecules, i.e., a subpopulation of “total”proBNP. We found and could establish that this subpopulation of totalproBNP termed “native proBNP” is detectable by specific bindingpartners, preferably by polyclonal and/or monoclonal antibodies.

The subpopulation termed native proBNP must not necessarily be a uniformpolypeptide fragment. The length of the native proBNP polypeptide(s) mayvary. Most of the molecules recognized in a sandwich immunoassay fornative proBNP are expected to represent N-terminal proBNP (1-76) orfragments thereof. Preferably an assay for native proBNP is set up in amanner appropriate for measurement of NT-proBNP fragments comprisingamino acids 10 through 66. The characteristic property of native proBNPis the presence of a native proBNP epitope as recognized by MAB 1.21.3.

In one embodiment, the present invention relates to an antibodyspecifically binding to native proBNP, wherein said antibodyspecifically binding to native proBNP is an antibody which, in terms ofthe values for proBNP as determined in patient samples, correlates withan r value of at least r =0.95 or above to MAB 1.21.3.

Based on the findings, disclosure, and deposits of the presentinvention, the skilled artisan will have no problem in assessing whetheran antibody is specifically binding to native proBNP or to total proBNP.MAB 1.21.3 is considered a prototype example of an antibody specificallybinding to native proBNP, whereas MAB 18.4.34 is considered a prototypeexample of an antibody to total proBNP. Any binding agent to proBNPwhatsoever can now be assessed for its binding specificity to native ortotal proBNP, respectively.

An antibody “specifically binding” to native proBNP is an antibodywhich, in terms of the values for proBNP as determined in patientsamples, correlates with an r value of at least r=0.95 or above to MAB1.21.3. Binding specificity to native proBNP is assessed in relation tothe binding properties of MAB 1.21.3 using relevant clinical samples. Atleast 20 and at most 25 serum samples obtained from patients with anNT-proBNP level of 10 ng/ml to 150 ng/ml of native proBNP are used.Binding to proBNP is determined with the BIACORE 3000 system. The valuesmeasured are correlated to native proBNP values as measured using MAB1.21.3, and the statistical assessment is performed by linear regressionanalysis. A linear regression of the type y=ax+b preferably is fittedusing Microsoft Excel, and the coefficient of correlation r and theslope is calculated. Even more preferred, such antibody will detectessentially the same native proBNP subpopulation of total proBNP asbound by MAB 1.21.3, wherein a binding to the essentially the samesubpopulation results in a correlation of r=0.98 or higher according tothe above procedures.

MAB 18.4.34 may be considered a prototype antibody for measurement oftotal proBNP. For any antibody specifically binding to native proBNP,using the same samples and procedures as described above, thecorrelation to MAB 18.4.34, i.e., to total proBNP, typically will belower as compared to the correlation to MAB 1.21.3. Preferably thecorrelation to MAB 18.4.34 for an antibody specifically binding to thenative proBNP subfraction of proBNP will be r=0.94 or below. Even morepreferred, it will be r=0.9 or below or as low as r=0.8 or below.

When comparing absolute amounts measured in such method comparisons, theassays detecting total proBNP consistently yield about 2- to 20-fold, inmost cases about 2- to 5-fold, higher values of proBNP as compared toMAB 1.21.3. In addition to the above specified correlation, a preferredantibody specifically binding to native proBNP will in the above methodcomparison also show a slope of less than 1.5. Most preferred, the slopewill be between 0.4 and 1.5.

The specific binding preferably occurs with a binding affinity of least10⁷ L/mol. The specific binding agent more preferred has an affinity of10⁸ L/mol or even more preferred of 10⁹ L/mol for native proBNP.

As explained above, a very important and preferred characteristic of MAB1.21.3 is the fact that only a variable fraction of between about 5% andabout 50% of total proBNP as comprised in a typical clinical sample isbound by this antibody.

A prototype example of an antibody which specifically binds to nativeproBNP is the monoclonal antibody as produced by clone MAB 1.21.3 whichhas been deposited with the DSMZ. MAB 1.21.3 is a sheep monoclonalantibody and has been produced as described in the Specific Embodimentssection. The epitope on proBNP recognized by these and by otherantibodies has been identified, characterized, and mapped by use ofshort synthetic peptides corresponding to well-defined sequences ofproBNP. This method is known and referred to as PepScan analysis.

In brief, 69 synthetic peptides comprising 8 consecutive amino acids ofproBNP have been synthesized comprising an N-terminal cysteine, a spacermolecule, and biotin. Each of these peptides was shifted by one aminoacid from the N- to the C-terminus. Peptide 1 thus comprises amino acids(aa's) 1 to 8, peptide 2, aa's from aa's 2 to 9, etc., and peptide 69,aa's 69 to 76.

MAB 1.21.3 has been found to significantly react with peptides number 39(amino acids 38-46) to 42 (aa's 42-49) which have the amino acidpositions 42 to 46 of proBNP in common. It therefore can be concludedthat MAB 1.21.3 reacts with an epitope essentially consisting of aminoacids 42 to 46 of proBNP.

As the skilled artisan appreciates, the presence or absence of anepitope may depend on tertiary structure, secondary modifications,complex formation, accessibility, and so on. Obviously, MAB 1.21.3 andother antibodies to native proBNP have very specific requirements inthis regard and do not react with the majority of proBNP-moleculespresent in a typical sample. Since the short synthetic PepScan peptidesare unlikely to have a tertiary structure or secondary modifications theepitope recognized by MAB 1.21.3 should be unmodified, and theterminology “native” has therefore been considered appropriate.

An assay based on an antibody to native proBNP and an assay measuringtotal proBNP exhibit striking differences in reaction intensity oncesynthetic proBNP (1-76) and proBNP as comprised in a clinical sample,like human serum, respectively, are measured and compared. Usingsynthetic proBNP (1-76), detection procedures can easily be set up andstandardized. Employing such assays, synthetic proBNP (1-76) either in asynthetic matrix or supplemented to a native sample, like human serum,is measured to the same levels in both these assays. Surprisingly,however, striking differences are found once proBNPas comprised in anative sample like human serum, is measured both these assays.

An assay employing an antibody or antibodies, respectively, reactive tototal proBNP, like monoclonal antibody MAB 17.3.1, 18.4.34, or 18.29.23,respectively, appears to detect all the proBNP molecules present in aserum sample, i.e., total proBNP. On the contrary, an assay based on anantibody reactive with native proBNP, e.g., MAB 1.21.3, only detects afraction of this total proBNP.

It can be demonstrated by the present invention that the native proBNPsubpopulation of total proBNP clinically shows a very good correlationto the biologically active BNP. Of course, in the measurement of BNP,all the precautions for obtaining correct BNP values with respect tosampling and handling have been observed. For measurement of nativeproBNP, routine sample processing without specific precautions provedsatisfactory.

All the data established with the present invention clearly indicatethat the epitope identified in the present invention and as specificallybound by MAB 1.21.3 is a major epitope for appropriate antibodies tonative proBNP. Obviously, this native proBNP epitope as recognized byMAB 1.21.3 can undergo a natural modification or can become part of aprotein complex which changes this epitope, with the effect that MAB1.21.3 binds to such modified or complexed proBNP to a lower extent ornot at all. The proBNP carrying such modified “non-native” proBNPepitope is only significantly measured in assays for total proBNP.

Because of their intrinsic high reproducibility, monoclonal antibodiesare preferred tools to detect native proBNP. In a preferred embodiment,the present invention therefore relates to a monoclonal antibodyspecifically binding to native proBNP.

As the skilled artisan will appreciate, other monoclonal antibodies maybe found which, compared to MAB 1.21.3, may show a slightly differentpattern in reactivity to PepScan peptides numbers 35 to 38. A monoclonalantibody to native proBNP will not depart from the spirit of thisinvention as long as only a subpopulation of total proBNP is detected,which correlates with an r value of at least r=0.95 or above to thenative proBNP subpopulation comprised in the total proBNP population andas bound by MAB 1.21.3. Such correlation is determined using the BIACOREsystem and the statistical assessment as described above. Even morepreferred, such monoclonal antibody will detect essentially the samenative proBNP subpopulation of total proBNP as bound by MAB 1.21.3,wherein the essentially the same subpopulation results in a correlationof r=0.98 or higher according to the above procedures.

In a preferred embodiment, the present invention also relates to amethod of producing a monoclonal antibody, the method comprising thesteps of immunizing an appropriate non-human animal with proBNP,preferably a mouse, a rat, a rabbit, or a sheep, obtaining B-cellsproducing antibodies thereto, fusing these B-cells to appropriate fusionpartners, and testing the antibodies produced by the hybridomas thusobtained for reactivity to native proBNP. Preferably only suchmonoclonal antibodies are selected and used in an immunoassay which inappropriate patient samples correlate to MAB 1.21.3 with an r value ofat least 0.95. Such correlation is assessed as described above.Preferably the immunization is performed with synthetic proBNP or aproBNP produced in a prokaryotic host or with a synthetic peptide orfragments of proBNP, both at least comprising amino acids 41 to 44 ofproBNP.

Now that MAB 1.21.3 is available, it is of course also possible toproduce, purify, and identify polyclonal antibodies which can be used inthe specific detection of native proBNP. It has, e.g., been found that apolyclonal antibody to native proBNP can now be generated, purified, andcharacterized by its correlation to MAB 1.21.3.

As the skilled artisan will appreciate, there are various ways toproduce a PAB which binds to native proBNP. Obviously it will, e.g., bepossible to use one or more synthetic peptides as an immunosorbent invarious successful routes of immunopurification.

Polyclonal antibodies to native proBNP will not depart from the spiritof this invention as long as only a subpopulation of total proBNP isdetected, which correlates with an r value of at least r=0.95 or aboveto the native proBNP subpopulation comprised in the total proBNPpopulation and as bound by MAB 1.21.3. Such correlation is determinedusing the BIACORE system and the statistical analysis as describedabove. Even more preferred, such polyclonal antibody preparation willdetect essentially the same native proBNP subpopulation of total proBNPas bound by MAB 1.21.3, wherein such binding of the essentially the samesubpopulation results in a correlation of r=0.98 or higher according tothe above procedures.

One way to obtain such PAB to native proBNP is to immunize withrecombinant or synthetically produced proBNP, to purify the nativeproBNP-specific antibodies therefrom by affinity purification, and toassess the polyclonal antibody thus obtained via patient samples asdescribed above.

In a preferred embodiment, the present invention therefore relates to amethod of producing polyclonal antibodies, the method comprising thesteps of immunizing an appropriate non-human animal with proBNP,obtaining polyclonal antibodies thereto, and testing the antibodies thusobtained for reactivity to native proBNP. Preferably only suchpolyclonal antibodies are selected and used in an immunoassay for nativeproBNP which in appropriate patient samples correlate to MAB 1.21.3 withan r value of at least 0.95. Such correlation is assessed as describedabove.

For any polyclonal antibody specifically binding to native proBNP (usingthe same samples and procedures), the correlation to MAB 18.4.34, i.e.,to total proBNP, will be significantly lower as compared to thecorrelation to MAB 1.21.3. Since polyclonal antibody preparations mayalways contain individual antibodies with different properties,preferably the correlation to MAB 18.4.34 for a polyclonal antibodyspecifically binding to the native proBNP subfraction of total proBNPwill be r=0.94 or below. Even more preferred, it will be as low as 0.9or below. Preferably polyclonal antibody preparations specificallybinding to native proBNP will correlate to MAB 1.21.3 with r=0.98 orabove and to MAB 18.4.34 with r=0.94 or below, or even more preferably,with r=0.9 or below to MAB 18.4.34.

Preferably the immunization for obtaining polyclonal antibodies tonative proBNP is performed with synthetic proBNP or a proBNP produced ina prokaryotic host or with a synthetic peptide or fragments of proBNP,both at least comprising amino acids 41 to 44 of proBNP.

In the course of our experiments, a large variety of immunologicalreagents has been produced, analyzed, combined in various sandwichassays formats, and used in the detection of proBNP. These variouscombinations of immunological reagents revealed that the majority ofassays appears to measure total proBNP.

The total proBNP assays investigated have been found to exhibit areasonable correlation to BNP, which goes hand-in-hand with reasonablediagnostic accuracy for diagnosis of heart failure, cf. e.g., Mair, J.supra.

It could, however, now be established that an assay only detectingnative proBNP, as compared to an assay detecting total proBNP, betterdifferentiates between patients in NYHA class 0 or I and patients inNYHA classes II, III, or IV, respectively. This also leads to animproved clinical discrimination of heart failure patients.

In a preferred embodiment, the present invention therefore relates to amethod for specific detection of native proBNP comprising the steps ofcontacting a sample suspected or known to contain proBNP with anantibody specifically binding to native proBNP under conditions allowingfor the formation of an antibody to native proBNP-native proBNP complexand detecting the complex formed. Preferably said method for specificdetection of native proBNP is used to differentiate NYHA stages 0 and Ifrom NYHA stages II, III, or IV.

The “antibody to native proBNP-native proBNP complex” may also simply betermed “antibody-native proBNP complex”.

The term “antibody” relates to mono- or polyclonal antibodies, chimeric,or humanized or other antibodies obtainable by genetic engineering, aswell as all antibody fragments known to the expert such as F(ab′)₂,Fab′, or Fab fragments. Other binding agents with appropriatespecificity for native proBNP can be used to substitute for antibodiesor antibody fragments. Only the specific binding to native proBNP, inanalogy to MAB 1.21.3, must be ensured.

As the skilled artisan appreciates, there are numerous ways to detectnative proBNP employing an antibody specifically binding thereto, whichall are described in detail in relevant textbooks (cf., e.g., Tijssen,P., Practice and Theory of Enzyme Immunoassays 11 (1990) Elsevier,Amsterdam, or Diamandis, et al., eds. (1996) Immunoassay, AcademicPress, Boston).

In the context of the present invention, many reagents and reagentcombinations for detection of total proBNP or native proBNP have beenanalyzed by the BIACORE system, some results of which are shown in theSpecific Embodiments section.

In clinical routine diagnostics, frequently methods based on aheterogeneous immunoassay format are used. In a preferred embodimentaccording to the present invention, the method for detection of nativeproBNP is a competitive immunoassay.

Even more preferred are immunoassays according to the sandwich assayprinciple, in which an antibody-antigen-antibody complex, also called asandwich, is formed.

In a preferred embodiment according to the present invention, the methodfor specific detection of native proBNP is a sandwich immunoassay,wherein a first antibody to native proBNP and a second antibody to totalproBNP are used, and wherein the second antibody to proBNP and the firstantibody to native proBNP both bind to native proBNP at differentepitopes, thus forming a (first) anti-native proBNP antibody-nativeproBNP-(second) anti-proBNP antibody complex.

As the skilled artisan will appreciate, a sandwich assay for detectionof native proBNP can also be set up using the antibody to total proBNPas a first (capture) antibody and the anti-native proBNP antibody as asecond (tracer, detection, or labelled) antibody.

Preferably, such a sandwich method for determination of the nativeproBNP comprises the following steps:

-   -   (a) mixing a sample with a first native proBNP-specific antibody        carrying a group suitable for binding to a solid phase or mixing        the sample with the first native proBNP-specific antibody which        is already bound to a solid phase,    -   (b) mixing this solution with a second antibody to total proBNP        binding to an epitope outside the native proBNP epitope, which        is present on both native proBNP and total proBNP, and carrying        a label under conditions that a first antibody-native        proBNP-second antibody complex is formed,    -   (c) binding the immune complex formed to a solid phase,    -   (d) separating the solid phase from the liquid phase, and    -   (e) detecting the label in one or both phases.

In a quantitative determination, the same measurement is carried outwith a defined amount of native proBNP as a standard, and after thedetermination of the sample, a step (f) is performed, i.e., themeasuring values of the standard or standard curve are compared to thoseobtained with the sample, and the corresponding concentration of nativeproBNP is extrapolated.

The first antibody specific for native proBNP can be bound directly tothe solid phase or indirectly via a specific binding pair system. Thedirect binding of this antibody to the solid phase follows methods knownto the expert, for example, in an adsorptive way. If the binding isindirect via a specific binding pair system, the first antibody is aconjugate consisting of an antibody against native proBNP and a firstpartner of the specific binding pair system. A “specific binding pairsystem” means two partners which can react specifically with each other.This binding can be based on an immunological binding or on analternative specific binding. Preferred combinations are biotin andavidin, streptavidin or anti-biotin, respectively, hapten andanti-hapten, Fc-fragment of an antibody and antibodies against thisFc-fragment, or carbohydrate and lectin. Preferably, a combination ofbiotin and avidin or of biotin and streptavidin is used as a specificbinding pair system.

The second partner of the specific binding pair system is coated to asolid phase. Streptavidin or avidin are used preferably. The binding ofthis partner of the specific binding pair system to an insoluble carriermaterial can be performed according to standard procedures known to theexpert. Here a covalent as well as an adsorptive binding is suitable.

As a solid phase, test tubes or microtiter plates made of polystyrene orsimilar plastics are suitable which are coated with the second partnerof the specific binding pair system. Further suitable and particularlypreferred are particulate substances such as latex particles, magneticparticles, molecular sieve materials, and glass corpuscles. Paper ornitrocellulose can also be used as carriers. Use of magnetic beadscoated with the second partner of the specific binding pair system asdescribed above is particularly preferred. After completion of theimmunological reaction and binding of the immunological complex formedto the solid phase, these microparticles can be separated from theliquid phase, for example, by filtration, centrifugation, or in the caseof the magnetic particles, via a magnet. Detection of label bound to thesolid phase, or of the label remaining in the liquid phase or of both,is then performed according to standard procedures.

The second antibody binding to total proBNP, binds to an epitope outsidethe native proBNP epitope which is present on both native proBNP andtotal proBNP. Simultaneous binding of both antibodies to these twoepitopes on the native proBNP molecule must be possible becauseotherwise, no sandwich complex would form.

The investigators of the present invention have also identified epitopeson proBNP which are very appropriate for the sandwich assay describedabove.

A large number of monoclonal antibodies has been generated. It could beestablished that not all epitopes recognized on recombinant proBNP areequally appropriate to measure proBNP in a patient sample.

Three epitopes essentially consisting of amino acids 13-16, 27-31, and64-67, respectively, as recognized by MAB's 17.3.1, 18.4.34, and18.29.23, respectively, appear to be present on the vast majority of(N-terminal) μproBNP molecules, i.e., are epitopes of total proBNP.These hybridomas have been deposited with the DSMZ on May 7, 2003. Theantibodies produced by these hybridomas represent ideal tools formeasurement of total proBNP. If used alone in a competitive assay formator in combination with each other or a PAB reacting with total proBNP ina sandwich assay format, total proBNP can be easily measured.

The preferred hybridoma cell lines according to the inventionMAB<NT-proBNP>16.1.39 (=MAK<NT-proBNP>16.1.39=MAB 16.1.39),MAB<NT-proBNP>17.3.1, MAB<NT-pro BNP>1.21.3, MAB<NT-proBNP>18.4.34, andMAB<NT-proBNP>18.29.23, were deposited, under the Budapest Treaty on theinternational recognition of the deposit of microorganisms for thepurposes of patent procedure, with Deutsche Sammlung von Mikroorganismenund. Zellkulturen GmbH (DSMZ), Germany, as follows: Cell line Depositionno. Date of deposit MAK<NT-proBNP>17.3.1 DSM ACC 2591 May 7, 2003MAK<NT-proBNP>18.4.34 DSM ACC 2592 May 7, 2003 MAK<NT-proBNP>18.29.23DSM ACC 2593 May 7, 2003 MAK<NT-proBNP>1.21.3 DSM ACC 2650 Apr. 27, 2004

The antibodies obtainable from said cell lines are preferred embodimentsof the invention.

In the detection of native proBNP, preferably a monoclonal antibody tototal proBNP as described above is used in a sandwich assay incombination with an antibody specifically binding to native proBNP. Suchsandwich then results in an assay specifically detecting only the nativeproBNP subpopulation of total proBNP. Preferred antibodies to totalproBNP in such sandwich for measurement of native proBNP are antibodiesessentially binding to amino acids 13-16, 27-31, and 64-67,respectively. These epitopes, for example, are recognized by MAB's17.3.1, 18.4.34, and 18.29.23, respectively. Most preferably an antibodybinding to amino acids 27 to 31, like MAB 18.4.34, is used in suchsandwich assay for native proBNP.

All biological liquids known to the expert can be used as a sample in amethod for specific detection of native proBNP in vitro. The preferredsamples for in vitro diagnosis are body fluids like whole blood, bloodserum, blood plasma, urine, or saliva. The use of serum or plasma,respectively, is particularly preferred.

Besides the so-called wet tests as described above with test reagents ina liquid phase, all standard dry test formats suitable for the detectionof antigens, haptens, peptides, proteins, antibodies, etc. can be usedtoo. These dry tests or test strips, as for instance described in EP186,799, combine all test components on one single carrier except thesample to be analyzed.

In a preferred embodiment, the present invention relates to a method fordiagnosing heart failure comprising detecting native proBNP andcorrelating the level of native proBNP to the presence of heart failure.As the skilled artisan will appreciate, the level of native proBNP canalso be used to assert the absence or the severity of heart failure.

It is also preferred to use a measurement of native proBNP in thefollow-up of patients with heart failure and in the monitoring oftreatment.

A further preferred embodiment relates to a kit for measurement ofnative proBNP comprising an antibody specifically binding to nativeproBNP and auxiliary reagents for detection of native proBNP.

The examples, references, sequence listing, and figures are provided toaid the understanding of the present invention, the true scope of whichis set forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

Specific Embodiments EXAMPLE 1 Method of Production of RecombinantN-Terminal proBNP (1-76)

Cloning of the Recombinant N-Terminal proBNP

The nucleotide sequence of the N-terminal proBNP (amino acid sequence1-76) was produced by means of genetic synthesis. To obtain an optimumexpression of the gene in E. coli, the DNA sequence was suited to thecodons most frequently used in E. coli. The sequences of theoligonucleotides used for the production of the gene are the following:Pro5′(SEQ ID NO:1): 5′CCGGATCCCACCCGCTG3′ ) Pro1hum (SEQ ID NO:2):5′CGGGATCCCACCCGCTGGGTTCCCCGGGTTCCGCTTCCGACCTGGAAACCTCCGGTCTGCAGGAACAGCGTAACCACCT3′ Pro2hum (SEQ ID NO:3):5′CGGTTCCAGGGAGGTCTGTTCAACCTGCAGTTCGGACAGTTTACCCTGCAGGTGGTTACGCTGTTCCTGC3′ Pro3hum (SEQ ID NO:4):5′CAGACCTCCCTGGAACCGCTGCAGGAATCCCCGCGTCCGACCGGTGTTTGGAAATCCCGTGAAGTTGCTAC 3′ Pro4hum (SEQ ID NO:5):5′CCCAAGCTTAACGCGGAGCACGCAGGGTGTACAGAACCATTTTACGGTGACCACGGATACCTTCGGTAGCAACTTCACGGGATTTCC3′ Pro3′ (SEQ ID NO:6):5′CCCAAGCTTAACGCGGAGC3′

The production of the gene was carried out with these primers usingpolymerase chain reaction (PCR). The amplified gene was cloned in asuitable vector like, for example, the vector pUC19 and then sequenced.For the cloning of the gene in the expression vector pQE8, the gene wascut out of the vector pUC19 via the restriction cutting points Bam Hiand Hind III and then ligated in the vector pQE8 allowing an expressionof proteins with N-terminal histidine tag and transformed in E. coli M15[pREP4].

Expression of the N-Terminal proBNP in E. coli

For the expression of the gene in E. coli , an over-night culture of arecombinant E. coli clone was transfected 1/60 in Luria broth (with 100μg/ml ampicillin and 50 μg/ml kanamycin) and induced at an OD 550 of 1with IPTG (isopropylthiogalactoside, 1 mM final concentration). Afterthe induction, the cultures were further incubated for 4 hours at 37° C.The cultures were then centrifuged and the cell pellet gathered in 50 mMNa phosphate buffer, pH 8.0, 300 mM NaCl. After decomposition of thecell suspension via ultrasound, the suspension was centrifuged and thesupernatant applied on a Ni-NTA (nitrile triacetate) column. After awashing step with 50 mM Na phosphate buffer, pH 8.0, 300 mM NaCl, 20 mMimidazole the histidine-tagged N-terminal proBNP was eluted with 50 mMNa phosphate buffer, pH 8.0, 300 mM NaCl, 300 mM imidazole. The elutedfractions were gathered and dialyzed against 50 mM Tris, pH 8.0. Toseparate impurities the dialysate was applied to a Q sepharose column.The mass of the purified N-terminal proBNP was determined via MALDI-TOF.This preparation (=recombinant proBNP) was found to contain proBNP 1-76and proBNP 1-66, the later most likely representing a degradationproduct.

EXAMPLE 2 Synthesis of NT-proBNP (1-76 amide)

NT-proBNP (1-76 amide) (SWISSPROT accession no. P16860, aa 27 to aa 134)was synthesized by an optimized solid phase peptide synthesis protocol(Merrifield (1962) Fed. Proc. Fed. Amer. Soc Exp. Biol. 21, 412) on anABI 433 peptide synthesizer. In brief, the peptide was assembled on aRink-linker modified polystyrene solid phase by repeatedly conjugatingan eightfold excess of amino acids each protected by temporarypiperidine labile Fmoc- and permanent acid labile tBu-, BOC-, OtBu-,Trt- or Pmc-groups depending on the side chain function. To get anoxidative stabile material the methionine at position 10 was exchangedby the equivalent amino acid norleucine. Further, to stabilize againstproteolytic degradation the C-terminus was amidated by using the Rinklinkage. After the assembly the fully protected peptide was removed fromthe solid phase and the permanent protecting groups were released bytreatment with trifluoracetic acid in a mixture of suitable cationscavengers and finally isolated by preparative reverse phase HPLCpurification. Three 125 μmol scale syntheses yielded 16.0, 17.1 and 18.0mg RP-HPLC single peak pure material (lyophilisate), respectively. Theidentity was proven by MALDI- and ESI-mass spectroscopy [8439.4].

EXAMPLE 3 Production of and Screening for Monoclonal Antibodies AgainstTotal or Native proBNP

Obtaining Monoclonal Antibodies Against N-Terminal proBNP

Balb/c mice, 8-12 weeks old, are subjected to intraperitonealimmunization with 100 μg N-terminal proBNP antigen, with completeFreund's adjuvant. Recombinant as well as proBNP (1-76) producedsynthetically by peptide synthesis, respectively, has been used as anantigen in mice. After 6 weeks three further immunizations are performedat 4-week intervals. One week after the last immunization blood is takenand the antibody titre is determined in the serum of the test animals.From the spleen of positively reacting mice, B-lymphocytes are obtainedand subjected to fusion with a permanent myeloma cell line. The fusionis carried out according to the well-known method of Köhler andMillstein (Nature 256, 1975, p. 495-497). The primary cultures of thepositive hybridomas are cloned in a usual way for example by using thecommercially available cell sorter or by “limiting dilution”.

For the production of ascites 5×10⁶ hybridoma cells areintraperitoneally injected in Balb/c mice which had been treated 1-2times with 0.5 ml Pristan before. After 2-3 weeks ascites liquid can beobtained from the abdominal region of the mice. From this, theantibodies can be isolated in the usual way.

Screening Test for Monoclonal Antibodies Against proBNP Peptides,Synthetic proBNP, and proBNP in Human Serum, Respectively

To identify the presence of antibodies against proBNP in the culturesupernatant of the hybridoma cells, supernatants are evaluated accordingto three screening assay formats.

Reactivity with Synthetic N-Terminal proBNP

Microtiter plates (Nunc, Maxisorb) are bound with 2.5 μg/ml syntheticNT-proBNP as an antigen in a loading buffer (Coating buffer, Cat. No.0726 559, Scil Diagnostics, GmbH) 100 μl/well, for 1 hour at roomtemperature under stirring. The post-loading is carried out in PBSbuffer (phosphate buffered saline, Oxid, Code-BR 14a) and 1% Byco C, for30 minutes. Subsequently, washing is performed with washing buffer (0.9msodium chloride solution, 0.05% TWEEN 20). The antibody sampleincubation is carried out with 100 μl/well for 1 hour at roomtemperature under stirring. A further washing step with washing solutiontakes place twice then. Afterwards, a further incubation is carried outwith the detection antibody PAB<M-Fcy>goat-F(ab′)₂-peroxidase conjugate(Chemicon, Cat. No. AQ127P), 100 mU/ml, 100 μl/well, for 1 hour at roomtemperature under stirring. After a further washing step with washingbuffer the peroxidase activity is established in the usual way, forexample, with ABTS, for 30 minutes at room temperature, and theextinction difference is read in mU at 405 nm by means of an ELISAreader.

Epitope Characterization using Synthetic Peptides for Epitope Analysis

For epitope analysis, streptavidin-loaded microtiter plates areincubated with peptide-biotin conjugates derived from the sequence ofproBNP (1-76). The complete proBNP sequence was scanned by applying 698-mer peptides shifted through the sequence in single amino acid steps,i.e., 1-8, 2-9, 3-10, 4-11 to 66-73, 67-74, 68-75, and 69-76,respectively. Additional biotinylated sequences have been testedcomprising the amino acid positions 1-10, 8-18, 1-21, 16-30, 30-38,32-43, 39-50, 47-57, 50-63, 62-70, and 64-76, respectively. Theindividual antigenic peptides have been dissolved to 250 ng/ml in PBSbuffer (phosphate buffered saline, Oxid, Code-BR 14a) with 0.5% Byco C.For peptide coating, 100 μl of each solution has been distributed indistinct wells of the microtiter plates which were then gently agitatedfor 1 hour under room temperature. Subsequently, washing was carried outwith washing buffer (0.9 m sodium chloride solution, 0.05% TWEEN 20).The antibody sample incubation and the detection reaction are performedas described above. Due to their reactivity with certain NT-proBNPpeptides, the position of the epitope as recognized by a mono- orpolyclonal antibody could be delineated.

Reactivity with proBNP in a Patient Sample

Wells of microtiter plates (Nunc, Maxisorb) are coated with 5 μg/mlPAB<human proBNP>S-IgG (IS, (1-21) or (30-38) S-IgG in loading buffer(Coating buffer, Cat.No. 0726 559, Scil Diagnostics, GmbH), 100 μl/well,for 1 hour at room temperature under stirring. The post-loading iscarried out in PBS buffer (phosphate buffered saline, Oxid, Code-BR 14a)and 1% Byco C, for 30 minutes. Subsequently, washing is performed withwashing buffer (0.9 sodium chloride solution, 0.05% TWEEN 20). Theincubation with native antigen in patient plasma, diluted in PBS buffer,is carried out with 100 μl/well for 1 hour at room temperature understirring. After a further washing step, the hybridoma supernatantincubation is performed with 100 μl/well for 1 hour at room temperatureunder stirring. Subsequently, washing is carried out twice with washingsolution and a further incubation is performed with the detectionantibody PAB<M-Fcy>Goat-F(ab′)₂-peroxidase conjugate (Chemicon, Cat. No.AQ127P), 100 mU/ml, 100 μl/well, for 1 hour at room temperature understirring. After a further washing step with washing buffer, theperoxidase activity is established in the usual way, for example, withABTS, for 30 minutes at room temperature, and the extinction differenceis read in mU at 405 nm by means of an ELISA reader.

Only those hybridoma cultures have been further processed which reactedpositively with synthetically produced N-terminal proBNP or with proBNPin human serum.

EXAMPLE 4 Production of Sheep Monoclonal Antibodies Against pro BNP

Obtaining Monoclonal Sheep Antibodies Against N-Terminal proBNP

Sheep were immunized with recombinant N-terminal proBNP in completeFreund's adjuvant. The dose was 0.1 mg per animal. The immunizationswere repeated at 4-week intervals in a period of 10 months. Six weeksafter the first immunization and afterwards once a month, the serumsamples were obtained and tested for their sensitivity and titer.

The lymphocytes for fusions were obtained from lymph nodes of immunizedsheep. Therefore 3 days before removal of the lymph node, a final boostinjection with recombinant N-terminal proBNP was done directly into aninguinal sheep lymph node.

After surgical removal of the lymph node, the lymphocytes were preparedand isolated under sterile condition. About 2×10⁹ cells from one lymphnode were stored in liquid nitrogen at a density of 1×10⁸ cells/vial.

For fusions, a vial of 1×10⁸ frozen lymph node lymphocytes were thawedand mixed with hypoxanthine and thymidine sensitivemyeloma/heteromyeloma cells (mouse NS1×sheep lymphocytes, clone 1 C 10,Bioventix, Inc.) in a ratio of 2:1 with fusing agent polyethylene glycol(PEG).

Several 96-well plates were seeded with 1-3×10⁴ cells (orheteromyelomas) per well and cultivated in selection medium. After 8-10days, examination and screening of the hybridoma cells for reactivitywith N-terminal pro BNP were done by ELISA assay.

The primary cultures of the positive hybridomas were cloned in the usualway by using the commercially available cell sorter or by “limitingdilution”.

Screening Test for Monoclonal Antibodies Against proBNP Peptides,Synthetic proBNP, and proBNP in Human Serum, Respectively

To identify the presence of antibodies against proBNP in the culturesupernatant of the hybridoma cells, supernatants were evaluatedaccording to three screening assay formats.

Reactivity with Synthetic N-Terminal proBNP

Microtiter plates (Nunc, Maxisorb) were coated with 2.5 μg/ml syntheticNT-proBNP as an antigen in a loading buffer (Coating buffer, Cat. No.0726 559, Scil Diagnostics, GmbH) 100 μl/well, for 1 hour at roomtemperature under stirring. The post-loading was carried out in PBSbuffer (phosphate buffered saline, Oxid, Code-BR 14a) and 1% Byco C, for30 minutes. Subsequently, washing was performed with washing buffer (0.9sodium chloride solution, 0.05% TWEEN 20). The antibody sampleincubation was carried out with 100 μl/well for 1 hour at roomtemperature under stirring. A further washing step with washing solutionthen took place twice. Afterwards, a further incubation was carried outwith the detection antibody peroxidase-conjugated AffiniPure DonkeyAnti-Sheep IgG (Dianova Code Number 713-035-147) diluted 1:40,000 in PBSbuffer, 100 μl/well, for 1 hour at room temperature under stirring.After a further washing step with washing buffe, the peroxidase activitywas established in the usual way, for example, with ABTS for 30 minutesat room temperature, the extinction difference was read in mU at 405 nmby means of an ELISA reader.

Epitope Characterization using Synthetic Peptides for Epitope Analysis

For epitope analysis, streptavidin-loaded microtiter plates wereincubated with peptide-biotin conjugates derived from the sequence ofproBNP(1-76).

The complete proBNP sequence was scanned by applying 69 8-mer peptidesshifted through the sequence in single amino acid steps, i.e., 1-8, 2-9,3-10, and 4-11 to 66-73, 67-74, 68-75, and 69-76, respectively.Additional biotinylated sequences were tested comprising the amino acidpositions 1-10, 8-18, 1-21, 16-30, 30-38, 32-43, 39-50, 47-57, 50-63,62-70, and 64-76, respectively. The individual antigenic peptides weredissolved to 250 ng/ml in PBS buffer (phosphate buffered saline, Oxid,Code-BR 14a) with 0.5% Byco C. For peptide coating, 100 μl of eachsolution was distributed in distinct wells of the microtiter plateswhich were then gently agitated for 1 hour under room temperature.Subsequently, washing was carried out with washing buffer (0.9 M sodiumchloride solution, 0.05% TWEEN 20). The antibody sample incubation andthe detection reaction were performed as described above. Due to theirreactivity with certain NT-proBNP peptides, the position of the epitopeas recognized by a mono- or polyclonal antibody could be delineated.

An example of a PepScan is shown in FIG. 1. The monoclonal antibodysecreted by hybridoma 1.21.3 most strongly reacts with peptides numbers36 through 38. This corresponds to an epitope consisting of at least theamino acids 38 through 43 (SEQ ID NO: 11) of proBNP. Since strongestreactivity clearly is seen with peptides 37 and 38, the shared epitopemay be considered to comprise amino acids 38-42.

Reactivity with proBNP in a Patient Sample

Wells of microtiter plates (Nunc, Maxisorb) were coated with 5 μg/mlMAB<human proBNP>M-18.4.34-IgG in loading buffer (Coating buffer, Cat.No. 0726 559, Scil Diagnostics, GmbH), 100 μl/well, for 1 hour at roomtemperature under stirring. The post-loading was carried out in PBSbuffer (phosphate buffered saline, Oxid, Code-BR 14a) and 1% Byco C, for30 minutes. Subsequently, washing was performed with washing buffer (0.9sodium chloride solution, 0.05% TWEEN 20). The incubation with nativeantigen in patient plasma, diluted in PBS buffer, was carried out with100 μl/well for 1 hour at room temperature under stirring. After afurther washing step, the hybridoma supernatant incubation was performedwith 100 μl/well for 1 hour at room temperature under stirring.Subsequently, washing was carried out twice with washing solution, and afurther incubation with the detection antibody Peroxidase-conjugatedAffiniPure Donkey Anti-Sheep IgG (Dianova Code Number 713-035-147),diluted 1:40 000 in PBS buffer, 100 mU/ml, 100 μl/well, for 1 hour atroom temperature under stirring. After a further washing step withwashing buffer, the peroxidase activity was established in the usualway, for example, with ABTS for 30 minutes at room temperature, theextinction difference read in mU at 405 nm by means of an ELISA reader.

Only those hybridoma cultures were further processed which reactedpositively with synthetically produced N-terminal proBNP or with proBNPin human serum.

EXAMPLE 5 Production of Polyclonal Antibodies Against N-Terminal proBNP

Immunization

Sheep were immunized with recombinant N-terminal proBNP (see Example 1)in complete Freund's adjuvant. The dose was 0.1 mg per animal. Theimmunizations were repeated at 4-week intervals in a period of 10months. Six weeks after the first immunization and afterwards once amonth, the serum samples were obtained and tested for their sensitivityand titer.

Purification of Polyclonal Antibodies from Sheep Serum

Starting from the raw serum of a sheep immunized with recombinantN-terminal proBNP, lipid components were removed by delipidation withaerosil (1.5%). Afterwards the immunoglobulins were separated byammonium sulfate precipitation (2 M). The dissolved precipitate wasdialysed against 15 mM KPO₄, 50 mM NaCl, pH 7.0, and chromatographed onDEAE sepharose. The IgG fraction (=PAB<NT-proBNP>S-IgG(DE)) was obtainedin the flow through.

Affinity Chromatography for the Production of Polyclonal AntibodiesSpecific for Total proBNP

For the affinity purification of polyclonal antibodies bindingspecifically to total proBNP (=PAB<NT-proBNP>S-IgG(IS, 1-21) or briefly,PAB<1-21>), the peptide HPLGSPGSASDLETSGLQEQR-C ((1-21)21-Cys, SEQ IDNO: 7) was used. The affinity matrix was produced by covalently binding1 mg of the peptide (1-21)21-Cys to 2 ml of maleimide activatedEAH-Sepharose 4B (Amersham Biosciences, Product No 17-0569-01).

With 10 ml of the affinity matrix, a column was packed and equilibratedwith 50 mM KPO₄, 150 mM NaCl, pH 7.5 (PBS). Two g ofPAB<NT-proBNP>S-IgG(DE) were applied to the column. The column waswashed with PBS and 20 mM KPO₄, 500 mM NaCl, 0.1% TRITON X-100 (Rohm &Haas), 0.5% Na deoxicholic acid, pH 7.5. The IgG specifically bound tothe affinity matrix was eluted with ImmunoPure Gentle Ag/Ab elutionbuffer (Pierce, Product N° 21013) and is referred to asPAB<1-21>. Theaffinity matrix was regenerated with 1 M propionic acid and conserved inPBS/NaN₃.

A similar procedure was applied to generate affinity purified polyclonalantibodies PAB<NT-proBNP>S-IgG(IS,30-38) or briefly, PAB<30-38>)specific for total proBNP (Karl, J. et al., WO 00/45176).

Affinity Chromatography for the Production of Polyclonal AntibodiesSpecific for Native proBNP

The polyclonal antibody to native proBNP(=PAB<NT-proBNP>S-IgG(IS,41-46), or briefly PAB<41-46>) was obtained bysequential affinity chromatography. In the same way as described above,3 individual peptides, CEUEU-SLEPLQE ((37-43)37-Cys, SEQ ID NO: 8),CEUEU-SPRPTGVW ((44-51)44-Cys, SEQ ID NO: 9) and C-EPLQESPRPTG((39-50)39-Cys, SEQ ID NO: 10) (EUEU merely functions as an extendedlinker for the peptide following behind) were used for the production of3 individual affinity matrices. PAB<NT-proBNP>S-IgG(DE) was firstapplied to the affinity matrix comprising peptide (37-43)37-Cys toremove all polyclonal antibody primarily binding to the NT-proBNPsequence 37-43. The flow through was then applied to the second affinitymatrix comprising peptide (44-51)44-Cys to capture polyclonal antibodiesprimarily binding to the NT-proBNP sequence 44-51. The bound antibodieswere eluted and collected as described above (=PAB<44-5 1>). Finally,the flow through of the second affinity purification was passed over thethird affinity matrix comprising peptide (39-50)39-Cys. The boundantibodies were eluted and collected as described above. As determinedby the method known and referred to as PepScan analysis, the elutedantibodies from the third affinity matrix are specific for epitopes inthe sequence 41-46 (=PAB<41-46>) which represent the remaining epitopesof the overlapping sequence between 37-43 and 44-51.

EXAMPLE 6 BIACORE Analysis of Monoclonal and Polyclonal Antibodies toproBNP

The specificity of monoclonal and polyclonal antibodies to nativeNT-proBNP was determined by surface plasmon resonance using a BIACORE3000 analyzer. All surface plasmon resonance measurements were performedat 25° C. using the BIACORE 3000 equipped with a research-grade CM5sensor chip. The running buffer was HBS (10 mM HEPES, 150 mM NaCl, 3.4mM EDTA and 0.005% P20 (=Polysorbat) at pH 7.4)

Immobilization of the Ligand PAB<NT-proBNP,1-21>S-IgG

The ligand which was used as capture antibody for total NT-proBNP wasimmobilized using amine-coupling chemistry. Before coupling, the sensorchip was preconditioned at a flow rate of 20 μl/min by 10 μl injectionsof 0.1% SDS, 50 mM NaOH, 10 mM HCl, and 100 mM phosphoric acid. Thesurfaces of all flow cells were activated for 5 min with a 1:1 mixtureof 0.1 M NHS (N-hydroxysuccinimide) and 0.1 M EDC(3-(N,N-dimethyl-amino)propyl-N-ethylcarbodiimide) at a flow rate of 20μl/min. The ligand at a concentration of 30 μg/ml in 10 mM sodiumacetate, pH 5.0, was injected in all 4 flow cells for 5 min. Thesurfaces were blocked with a 5 min injection of 1 M ethanolamine, pH8.0, followed by 30 s injections of HBSwash (100 mM HEPES, pH 7.4, 1.5 MNaCl, 3.4 mM EDTA, 0.05% P20 (=Polysorbat), 2% DMSO), 100 mM HCl, and2×100 mM phosphoric acid to remove noncovalently bound ligand. Thedensity of the ligand was about 16,000 RU.

Concentration Measurements of NT-proBNP in Patient Samples

To perform the following method in BIACORE 3000, the program shown inFIG. 2 was used. Synthetic NT-proBNP(1-76)amid in concentrations of 0,2.5, 5, 10, 20, and 40 nM in 20% horse serum (horse serum diluted 1:5with HBS+1 mg/ml carboxymethyldextran) was used as calibrator. Theaddition of carboxymethyldextran was used to suppress non-specificbinding of serum components to the surface of the sensor chip).

Patient samples with >10 ng/ml native NT-proBNP were diluted 1:5 withHBS also containing 1 mg/ml carboxymethyldextran.

Calibrator and patient samples were injected at a flow rate of 10 μl/minfor 10 min over all four flow cells followed by a 30 s injection of HBSat a flow rate of 100 μl/min to remove non-specifically bound serumcomponents. The antibody whose specificity had to be determined wasinjected in a concentration of 500 nM in HBS for 3 min at a flow rate of10 μl/min, antibody 1 in flow cell 1, antibody 2 in flow cell 2, and soon. The binding data of the antibodies in RU were determined asdifference between the response 10 s before the injection of an antibodyand the response 10 s before the injection of the next antibody or HBS,respectively.

For the calculation of the NT-proBNP concentrations in the patientsamples, BIA evaluation software v. 4.1 was used. For each antibody, acalibration curve of synthetic NT-proBNP(1-76)amid was generated using aspline fit, and the corresponding concentrations of the 1:5 dilutedpatient samples were calculated. The concentrations were multiplied by 5to get the concentrations of NT-proBNP in undiluted sera.

Determination of the Specificity of an Antibody

In order to determine if an antibody binds to native or total NT-proBNPin human serum, the concentrations of NT-proBNP determined with theantibody in question (y-axis) were plotted against the concentrations ofthe corresponding sample determined with the reference antibody MAB1.21.3 (x-axis). A linear regression curve of the type y=ax+b was fittedusing Microsoft Excel and the coefficient of correlation r and the slopewere calculated. TABLE 1 Characteristics of various anti-proBNPantibodies Synthetic Patient sample Antibody Epitope recognized proBNPproBNP MAB 17.3.1 amino acids 13-16 +++ +++ MAB 18.4.34 amino acids27-31 +++ +++ MAB 18.29.23 amino acids 62-76 +++ +++ MAB 1.21.3 aminoacids 42-46 +++ + PAB <1-21> amino acids 1-21 +++ +++ PAB <44-51> aminoacids 44-51 +++ ++ PAB <41-46> amino acids 41-46 +++ ++++ indicates that both synthetic proBNP and proBNP in a patient sampleare recognized very well and to a similar extend+ indicates a reaction in the range of 15% with proBNP in a patientsample as compared to the value obtained with synthetic proBNP

From Table 1, it is readily evident that the vast majority of proBNPepitopes appears to be present on synthetic proBNP and proBNP ascomprised in a patient sample in the same manner. This is exemplified byMAB 17.3.1, MAB 18.4.34, MAB 28.29.13, and PAB<1-21>, respectively.

One epitope, however, appears not to be present on synthetic proBNP andproBNP as comprised in a patient sample in the same manner. This epitopeessentially consists of amino acids 41-44 and is recognized by MAB1.21.3 as well as by PAB<41-46>. It appears that, using theseimmunological reagents, only a subpopulation of the total proBNP aspresent in a patient sample is recognized. This leads to strikinglydifferent results when measuring proBNP in a patient sample with anassay for total proBNP or an assay for native proBNP, respectively. Onlythis subpopulation of total proBNP appears to carry an epitopecharacteristic for native proBNP.

As is obvious from FIGS. 3 to 8, all antibodies to native proBNP, i.e.,MAB 16.1.39 and PAB<41-46>, show a very good correlation to MAB 1.21.3,whereas the antibodies to total proBNP, i.e., MAB 18.4.34, MAB 18.29.23,and PAB 30-38, show a much lower correlation to MAB 1.21.3.PAB<44-51>interestingly appears to be of somewhat mixed reactivity andwould not qualify as an antibody specifically binding to native proBNPbecause it correlates to less than r=0.95 to MAB 1.21.3.

EXAMPLE 7 Clinical Comparison of Assays for Native and Total proBNP

In a clinical study, 246 patient samples classified according to theirNYHA status have been analyzed by sandwich immunoassays for nativeproBNP and total proBNP, respectively. The results of this study aregiven in Table 2. TABLE 2 Comparative analysis of native proBNP andtotal proBNP in patient samples Native proBNP Total arbitrary NYHAproBNP NYHA NYHA n 246 units X/NYHA 0 pg/ml X/NYHA 0 0 119 337 1.0 6381.0 1 32 355 1.1 717 1.1 2 62 655 1.9 1072 1.7 3 30 2947 8.7 3609 5.6 43 12755 38 15902 25

Clinically very important is the differentiation of patients with no orvery mild disease (HYHA classes 0 and 1) as compared to patients withdisease progression (NYHA X=classes 2 or more). As can be seen fromTable 2, there is a significant increase from class 0/1 to class 2 andhigher classes. This increase for all the classes 2, 3, and 4 is morepronounced for native proBNP as compared to total proBNP. Thistranslates to a better sensitivity/specificity profile and clinicalutility for native proBNP as compared to total proBNP.

1. An antibody that binds specifically to native proBNP, wherein saidantibody, in terms of values for proBNP as determined in patientsamples, has a coefficient of correlation of at least 0.95 or above toMAB 1.21.3.
 2. The antibody of claim 1, wherein the antibody is amonoclonal antibody.
 3. The antibody of claim 2, wherein the antibody isproduced by hybridoma cell line MAB 1.21.3.
 4. The antibody of claim 1,wherein the antibody is an isolated polyclonal antibody.
 5. A method forspecific detection of native proBNP comprising the steps of contacting asample suspected or known to contain proBNP with an antibody specificfor native proBNP, wherein the antibody, in terms of values for proBNPas determined in patient samples, has a coefficient of correlation of atleast 0.95 or above to MAB 1.21.3, under conditions allowing forformation of an antibody to native proBNP-native proBNP complex, anddetecting the complex formed.
 6. The method according to claim 5,wherein the detection is performed by a competitive immunoassay.
 7. Themethod according to claim 5, wherein the detection is performed by asandwich immunoassay wherein a second antibody to proBNP is also usedand wherein the second antibody to proBNP and the antibody to nativeproBNP both bind to native proBNP, thereby forming an antibody toproBNP-native proBNP-antibody to native proBNP complex.
 8. A method fordiagnosing heart failure comprising detecting specifically native proBNPin a patient sample and correlating the level of native proBNP detectedto heart failure.
 9. A kit for measurement of native proBNP comprisingan antibody and auxiliary reagents for detection of native proBNP,wherein the antibody, in terms of values for proBNP as determined inpatient samples, has a coefficient of correlation of at least 0.95 orabove to MAB 1.21.3.
 10. The hybridoma cell line MAB 1.21.3 as depositedwith Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH.